A significant motivation of the BRAIN Initiative is the desire to understand information processing in neuronal tissues in situ. Towards this end a steadily growing number of neuroscience investigators are turning to optical methods to monitor neuronal activity as opposed to more traditional methods that rely on electrodes. The most commonly used method involves genetically encoded, fluorescent calcium indicators (i.e. GCaMPs) combined with multiphoton or wide field microscopy. These methods allow activity measurements in large numbers of identified neurons from intact animals. Advancement of these studies rely on the development of improved, genetically encoded, protein-based indicators. Fluorescent intracellular calcium indicators produce robust signals in response to neuronal activity, however they i) exhibit very slow kinetics relative to action potentials, ii) have poor individual action potential reporting fidelity and iii) cannot enable visualization of hyperpolarizations of membrane potential. Fluorescence voltage indicators provide signals which are richer in information, more temporally relevant and offer a more direct measure of neuronal electrical activity. A number of new and more practically useful fluorescent voltage indicators have been developed over the past decade with improved properties. During a previous funding period our laboratory developed a high throughput workflow to create and screen protein-based, voltage sensitive indicators. Using this platform we have discovered several novel indicator templates and new indicators. With the steady increase in throughput of our screening workflow we have seen, as expected, more rapid improvements in indicator properties which has translated to greater practical use. However, the protein design and modification space is enormous hence we propose further scaling up of the screening throughput and widening the template design space. We propose to screen upwards of 1150 novel constructs per day or ~8000 per week. We will include random mutagenesis in the process given the newly developed screening capacity. With this new platform we are seeking to develop : i) indicators with maximum total ?burst? photon output in response to action potentials, ii) fluorescence output increases from a weak resting level lasting of between 2-40 ms in response to action potential-type voltage transients, iii) positive voltage/fluorescence output slope relationship indicators, iv) indicators with green, red and near IR emission spectrum, v) indicators targeted to subcellular regions of the neuron, and vi) probes with reduced bleach rates.